Comparative Analysis of Soybean Root Proteome Reveals Molecular Basis of Differential Carboxylate Efflux under Low Phosphorus Stress

Abstract

Carboxylate efflux from roots is a crucial and differential response of soybean genotypes to low phosphorus (P) stress. Exudation of carboxylic acids including oxalate, citrate, succinate and fumarate was induced under low P stress, particularly in P-efficient soybean genotypes. Enhancement of root length, surface area and volume further improved P acquisition under low P stress. To understand the molecular basis of carboxylate efflux under low P stress, the root proteome of contrasting genotypes (P-efficient: EC-232019 and P-inefficient: EC-113396) was compared. Among a total of 325 spots, 105 (32%) were differentially abundant proteins (DAPs) between sufficient (250 µM) and low P (4 µM) levels. Abundance of 44 (14%) proteins decreased by more than two-fold under low P stress, while 61 (19%) proteins increased by more than two-fold. Protein identification and annotation revealed that the DAPs were involved in a myriad of functions including carboxylic acid synthesis, carbohydrate, protein and lipid metabolism. Proteins with significant abundance included malate dehydrogenase, isocitrate dehydrogenase, phosphoglucomutase, phosphoglycerate mutase, fructokinase, enolase, phosphoglycerate kinase, triosephosphate isomerase, alcohol dehydrogenase, glucan water dikinase, glutamine synthetase and argininosuccinate lyase. Inferences from proteomic analysis suggests the crosstalk between various metabolic pathways implicated in conferring superior P acquisition efficiency under stress.

Keywords: Glycine max; carboxylate efflux; gene expression analysis; mass spectrometry; phosphorus stress; root proteome; two-dimensional gel electrophoresis.

Figure 1

Variation in (A) total biomass, (B) root-to-shoot ratio, (C) root length, (D) root surface area, (E) root volume, (F) root phosphorus (P) concentration (G) shoot P concentration and (H) total P uptake in contrasting soybean genotypes grown at sufficient (250 μM) and low (4 μM) P. Data correspond to mean ± Standard Error (n = 9). DW: dry weight. *, ** and *** denote significance at 0.05, 0.01 and 0.001 probability levels, respectively.

Figure 1

Variation in (A) total biomass, (B) root-to-shoot ratio, (C) root length, (D) root surface area, (E) root volume, (F) root phosphorus (P) concentration (G) shoot P concentration and (H) total P uptake in contrasting soybean genotypes grown at sufficient (250 μM) and low (4 μM) P. Data correspond to mean ± Standard Error (n = 9). DW: dry weight. *, ** and *** denote significance at 0.05, 0.01 and 0.001 probability levels, respectively.

Figure 2

Variation in root exudation of (A) oxalate, (B) malate, (C) citrate, (D) fumarate, (E) succinate, (F) pyruvate and (G) lactate in contrasting soybean genotypes grown at sufficient (250 μM) and low (4 μM) P. Data correspond to mean±SE (n = 9). “nd” denotes peak “not detectable”. RFW: Root fresh weight. *, ** and *** denote significance at 0.05, 0.01 and 0.001 probability levels, respectively.

Figure 3

(A) Venn diagram showing number of differentially abundant proteins on two-dimensional electrophoretic gels at low P condition in comparison to sufficient P. Number indicated in green color are decreased proteins at low P, while red color are increased proteins at low P. Numbers within parenthesis correspond to proteins with significant Mascot score. Distribution of differentially abundant proteins based on gene ontology with respect to (B) biological process and (C) cellular localization.

Figure 4

Root proteins increased by more than two-fold at low (4 μM) P in contrasting soybean genotypes. Arrows denote the protein spots zoomed for better visualization. Bars in the graph denote the mean (n = 3) normalized intensity of protein spots on gels of EC-232019 Sufficient P (orange, SP ER), EC-232019 Low P (red, LP ER), EC-113396 Sufficient P (violet, SP IENR) and EC-113396 Low P (green, LP IENR). Proteins with significant Mascot score are presented here. Refer Table 1 for entire list of increased proteins.

Figure 4

Root proteins increased by more than two-fold at low (4 μM) P in contrasting soybean genotypes. Arrows denote the protein spots zoomed for better visualization. Bars in the graph denote the mean (n = 3) normalized intensity of protein spots on gels of EC-232019 Sufficient P (orange, SP ER), EC-232019 Low P (red, LP ER), EC-113396 Sufficient P (violet, SP IENR) and EC-113396 Low P (green, LP IENR). Proteins with significant Mascot score are presented here. Refer Table 1 for entire list of increased proteins.

Figure 5

Root proteins decreased by more than two-fold at low (4 μM) P in contrasting soybean genotypes. Arrows denote the protein spots zoomed for better visualization. Bars in the graph denote the mean (n = 3) normalized intensity of protein spots on gels ofEC-232019 Sufficient P (orange, SP ER), EC-232019 Low P (red, LP ER), EC-113396 Sufficient P (violet, SP IENR) and EC-113396 Low P (green, LP IENR). Proteins with significant Mascot score are presented here. Refer Table 2 for entire list of decreased proteins.

Figure 5

Root proteins decreased by more than two-fold at low (4 μM) P in contrasting soybean genotypes. Arrows denote the protein spots zoomed for better visualization. Bars in the graph denote the mean (n = 3) normalized intensity of protein spots on gels ofEC-232019 Sufficient P (orange, SP ER), EC-232019 Low P (red, LP ER), EC-113396 Sufficient P (violet, SP IENR) and EC-113396 Low P (green, LP IENR). Proteins with significant Mascot score are presented here. Refer Table 2 for entire list of decreased proteins.

Figure 6

Relative transcript levels of genes encoding differentially abundant proteins (A) up-regulated and (B) down-regulated by more than two-fold at low (4 μM) P in soybean roots. Data correspond to mean±SE (n = 9). Spot ID corresponds to the protein spots in Supplemental Figure S3B,D. *, ** and *** denote significance at 0.05, 0.01 and 0.001 probability levels, respectively.

Figure 7

Model depicting involvement of several enzymes in carboxylic acid synthesis and glycolytic by-pass pathways functioning under low P stress in soybean roots. Carboxylic acids indicated in orange boxes were detected in the root exudates of soybean. Enzymes denoted in red boxes are differentially abundant under low P stress, relative fold change at low P compared to sufficient P in contrasting genotypes are denoted in red (EC-232019) and green (EC-113396) bars. Enzymes denoted in blue boxes might have a possible role in increasing carboxylic acid synthesis or glycolytic by-pass reactions under P stress. ADH: Alcohol dehydrogenase, ASL: Argininosuccinate lyase, CS: Citrate synthase, CYS: Cysteine synthase, E: Enolase, F: Fumarase, FK: Fructokinase, GDH: Glutamate dehydrogenase, GOGAT: Glutamate oxoglutarate amino transferase, GS: Glutamine synthetase, GWD: α-glucan water dikinase, ICDH: Isocitrate dehydrogenase, LDH: Lactate dehydrogenase, MDH: Malate dehydrogenase, ME: Malic enzyme, MS: Methionine synthase, NP-G3PD: Non-phosphorylating NADP dependent glyceraldehyde-3-phosphate dehydrogenase, OAT: Oxaloacetase, PEPC: Phosphoenolpyruvate carboxylase, PGK: Phosphoglycerate kinase, PGLD: Phosphogluconate dehydrogenase, PGLM: Phosphoglucomutase, PGM: Phosphoglycerate mutase, PK: Pyruvate kinase, PPDK: Pyruvate phosphate dikinase, SAHS: S-adenosyl homocysteine synthase, ScoAS: Succinyl coenzyme A synthetase, SDH: Succinate dehydrogenase, TPI: Triosephosphate isomerase. PEPC, CS and PK data derived from enzyme activity (data published in Vengavasi et al. [26]).